In the 1920s, shortly after the discovery of bacterial viruses (bacteriophages), the medical community began to extensively pursue the treatment of bacterial diseases with bacteriophage therapy. The idea of using phage as a therapy for infectious bacterial diseases was first proposed by d'Herelle in 1918, as a logical application of the bacteriophages' known ability to invade and destroy bacteria. Although early reports of bacteriophage therapy were somewhat favorable, with continued clinical usage it became clear that this form of therapy was inconsistent and unpredictable in its results. Disappointment with phage as a means of therapy grew, because the great potential of these viruses to kill bacteria in vitro was not realized in vivo. This led to a decline in attempts to develop clinical usage of phage therapy, and that decline accelerated once antibiotics began to be introduced in the 1940s and 50s. From the 1960s to the present, some researchers who adopted certain bacteriophages as a laboratory tool and founded the field of molecular biology have speculated as to why phage therapy failed.
Despite the general failure of phage as therapy, isolated groups of physicians have continued to try to use these agents to treat infectious diseases. Many of these efforts have been concentrated in Russia and India, where the high costs of and lack of availability of antibiotics continues to stimulate a search for alternative therapies. See for example Vogovazova et al., “Effectiveness of Klebsiella pneumoniae Bacteriophage in the Treatment of Experimental Klebsiella Infection”, Zhurnal Mikrobiologii, Epidemiologii Immunobiologii, pp. 5-8 (April, 1991); and Vogovazova et al., “Immunological Properties and Therapeutic Effectiveness of Preparations of Klebsiella Bacteriophages”, Zhurnal Mikrobiologii, Epidemiologii Immunobiologii, pp. 30-33 (March, 1992)]. These articles are similar to most of the studies of phage therapy, including the first reports by d'Herelle, in that they lack many of the controls required by researchers who investigate anti-infectious therapies. In addition, these studies often have little or no quantification of clinical results. For example, in the second of the two Russian articles cited above, the Results section concerning Klebsiella phage therapy states that “Its use was effective in . . . ozena (38 patients), suppuration of the nasal sinus (5 patients) and of the middle ear (4 patients) . . . In all cases a positive clinical effect was achieved without side effects from the administration of the preparation”. Unfortunately, there were no placebo controls or antibiotic controls, and no criteria were given for “improvement”.
Another clinical use of phage that was developed in the 1950s and is currently still employed albeit to a limited extent, is the use of phage lysate, specifically staphphage lysate (SPL). The researchers in this field claim that a nonspecific, cell-mediated immune response to staph endotoxin is an integral and essential part of the claimed efficacy of the SPL. [See, eg., Esber et al., J. Immunopharmacol., Vol. 3, No. 1, pp. 79-92 (1981); Aoki et al., Augmenting Agents in Cancer Therapy (Raven, N.Y.), pp. 101-112 (1981); and Mudd et al., Ann. NY Acad. Sci., Vol. 236, pp. 244-251 (1974).] In this treatment, it seems that the purpose of using the phage is to lyse the bacteria specifically to obtain bacterial antigens, in a manner that those authors find preferential to lysing by sonication or other physical/chemical means. Here again, some difficulties arise in assessing these reports in the literature, because, in general, there are no placebo controls and no standard antibiotic controls against which to measure the reported efficacy of the SPL. More significantly, there is no suggestion in these articles to use phage per se in the treatment of bacterial diseases. Moreover, the articles do not suggest that phage should be modified in any manner that would delay the capture/sequestration of phage by the host defense system.
Since many patients will recover spontaneously from infections, studies must have carefully designed controls and explicit criteria to confirm that a new agent is effective. The lack of quantification and of controls in most of the phage reports from d'Herelle on makes it difficult if not impossible to determine if the phage therapies have had any beneficial effect.
As there are numerous reports of attempts at phage therapy, one would assume that had it been effective, it would have flourished in the period before antibiotics were introduced. But phage therapy has been virtually abandoned, except for the isolated pockets mentioned above.
As noted above, some of the founders of molecular biology who pioneered the use of specific phages to investigate the molecular basis of life processes have speculated as to why phage therapy was not effective. For example, G. Stent in his book Molecular Biology of Bacterial Viruses, WH Freeman & Co. (1963) pp. 8-9, stated the following:                “Just why bacteriophages, so virulent in their antibiotic action in vitro, proved to be so impotent in vivo, has never been adequately explained. Possibly the immediate antibody response of the patient against the phage protein upon hypodermic injection, the sensitivity of the phage to inactivation by gastric juices upon oral administration, and the facility with which bacteria acquire immunity or sport resistance against phage, all militated against the success of phage therapy.”        
In 1973, Dr. Carl Merril discovered along with his co-workers that phage lambda, administered by various routes (per os, IV, IM, IP) to germ-free, non-immune mice, was cleared out of the blood stream very rapidly by the organs of the reticulo-endothelial system, such as the spleen, liver and bone marrow. [See Geier. Trigg and Merril, “Fate of Bacteriophage Lambda in Non-Immune, Germ-Free Mice”, Nature, 246, pp. 221-222 (1973).] These observations led Dr. Merril and his co-workers to suggest (in that same Nature article cited above) overcoming the problem by flooding the body with colloidal particles, so that the reticulo-endothelial system would be so overwhelmed engulfing the particles that the phage might escape capture. Dr. Merril and his co-workers did not pursue that approach at the time as there was very little demand for an alternative antibacterial treatment such as phage therapy in the 1970s, given the numerous and efficacious antibiotics available.
Subsequently, however, numerous bacterial pathogens of great importance to mankind have become multidrug resistant (MDR), and these MDR strains have spread rapidly around the world. As a result, hundreds of thousands of people now die each year from infections that could have been successfully treated by antibiotics just 4-5 years ago. [See e.g. C. Kunin, “Resistance to Antimicrobial Drugs—A Worldwide Calamity”, Annals of Internal Medicine, 1993;118:557-561; and H. Neu, “The Crisis in Antibiotic Resistance”, Science 257, 21 Aug. 1992, pp. 1064-73.] In the case of MDR tuberculosis, e.g., immunocompromised as well as non-immunocompromised patients in our era are dying within the first month or so after the onset of symptoms, despite the use of as many as 11 different antibiotics.
Medical authorities have described multidrug resistance not just for TB, but for a wide variety of other infections as well. Some infectious disease experts have termed this situation a “global crisis”. A search is underway for alternative modes and novel mechanisms for treating these MDR bacterial infections.
Bacteriophage therapy offers one possible alternative treatment. Learning from the failure of bacteriophage therapy in the past, the present inventors have discovered effective ways to overcome the major obstacles that were the cause of that failure.
One object of the present invention is to develop a drug delivery vehicle wherein bacteriophages are protected by attaching to their surfaces a substance that can mask the phage surface antigens. This masking can be achieved by means such as, but not limited to, attaching substances in close proximity to the antigenic site, or attaching substances directly into the antigenic site, in either case, thereby blocking the host defense system's components from making contact with the antigenic site. The purpose of masking the antigenic site is to enable a bacteriophage to delay being inactivated by the host defense system.
Substances which can be used to mask phage surface antigens include a variety of polymers, both synthetic and natural, including but not limited to: polyethers, such as polyethylene glycol, polypropylene glycol, polypropylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polybutanediol, polysaccharides, hyaluronic acid, collagen, albumin, dextran, carboxymethylcellulose, and poly-D,L-amino acids.
Polyethylene glycol (PEG) is a well established immune system modifier already in clinical use, one of its major properties being its ability to protect the antigenic sites of proteins from interaction with the immune system.
PEG adducts are known in the art to prolong the circulating life of proteins that interact with the HDS. [See e.g. Nucci, M. L. et al., The Therapeutic Value of Poly(ethylene glycol)-Modified Proteins, Advanced Drug Delivery Reviews, 6, 1991, 133-151]. In this way, a shell of PEG molecules around one or more of the antigenic proteins of the phage will sterically hinder those proteins from interacting with complement, with immune cells, or with any other aspect of the HDS.
One example of the use of PEG to sterically prevent the interaction of the HDS with the antigens of a protein, is the drug ADAGEN™ (pegademase bovine). This PEG-modified protein is currently marketed for the treatment of severe combined immunodeficiency disease (SCID), a disease which is associated with adenosine deaminase deficiency. PEGylation of the enzyme slows its degradation and thereby renders it efficacious as a therapeutic. [See e.g. Hershfield, M. et al, Treatment of Adenosine Deaminase Deficiency with Polyethylene Glycol-Modified Adenosine Deaminase, New England Journal of Medicine, 316:589-596 Mar. 5, 1987.] One of the derivatives of PEG that is reported to have great stability, as well as high affinity and selectivity as a linker to antigens that are to be masked, is monomethoxypoly(ethylene glycol) (mPEG).
There are a number of methods known in the art to activate polymers so that they will bind with the target protein. The reagents to be used in the present invention, and known in the art for the activation of polymers for binding, include: trichloro-s-triazine (cyanuric chloride); carbonyldiimidazole; succinic anhydride; and succinimidyl carbonate. Succinimidyl carbonate is preferred for use in the present invention. The adduct targets on the protein include (but are not limited to): specific amino acid groups, sulfhydryl groups, and or other applicable moieties of the phage surface antigens that are to be masked.
The physico-chemical alteration of bacteriophage in the present invention allows a delay in their inactivation by the HDS, so that the phage are no longer prevented by the HDS from reaching and killing the target bacteria. The masking of the phage antigenic sites also decreases the tendency for the human or animal recipient of the phage therapy to form antibodies against the phage. As a result, the phage therapy remains useful for longer periods of time, and/or for more courses of treatment.
In the present invention, the adduct of the polymer with phage surface proteins is custom designed by methods known in the art, e.g. by: 1) varying the molecular weight of the polymer, 2) altering the reaction variables, for example: the concentrations of the reagents (such as the molecule used to activate the PEG reaction); the time course of the reaction (this changes the percentage of the amino acid groups of the phage surface antigen that become modified); the temperature; the pH; etc., 3) altering the type of PEG activator being used; and/or 4) altering the PEG derivative chosen for the reaction—one example among many being the bifunctional analog of SC-PEG known as poly(ethylene glycol)-bis-N-succinimidyl carbonate (“BSC-PEG”). These alterations provide a variety of physico-chemically altered bacteriophages, from which the ones demonstrating the best ability to delay inactivation by the HDS can be selected.
Given that the chemical substances linked to the bacteriophages cannot be genetically transmitted to progeny, it follows that the daughter phages will have no protection (by physico-chemical alteration) from the HDS. Nevertheless for each physico-chemically altered bacteriophage that does succeed in infecting a bacterium, on average a few hundred daughter phages are released within about a half hour (the actual number released and the time to burst depend on factors including the strain of bacteria and the strain of phage). Many of these daughter phage then have the opportunity to infect nearby bacteria, before the HDS has time to inactivate them. Therefore the rate at which the phage are multiplying is greater than the rate at which they are being taken out (by phagocytosis, complement fixation, or any other aspect of the host defense system), resulting in exponential growth in the number of phages at the site of an infection. Thus the physico-chemically altered phage of the present invention establish a “beachhead”, wherein the succeeding “waves” of bacteriophage (the first “wave” being the parent generation of physico-chemically altered phage, and the following “waves” being the succeeding generations of unmodified daughter phages) combine to substantially eliminate the infectious bacteria.
While PEG and the other polymers listed above are examples of substances which can protect proteins from interaction with the HDS, there are many other suitable substances. Such substances are known to those skilled in the art and any of these substances can be used in the present invention.
Another object of the present invention is to develop a method for treating bacterial infectious diseases in an animal by administering to the animal, by an appropriate route of administration, an effective amount of the physico-chemically altered bacteriophage.